Contratto di ricerca Miguel Servet (ISCIII CD14 / 00032) a (GN-G.). Fellowship dalla Organizzazione olandese per la ricerca scientifica &quot;Graduate School of Translational Neuroscience Programma&quot; (022005019) a (CH). Le sovvenzioni da parte Prinses Beatrix Fonds (Progetto WAR08-12) e dell&#39;Association Française contre les Myopathies a (PM-M.); nonché da un Veni Fellowship dell&#39;Organizzazione olandese per la ricerca scientifica (916.10.148) una borsa di studio della Fondazione cervello dei Paesi Bassi (FS2008 (1) -28) e il Prinses Beatrix Fonds (Progetto WAR08-12) (a ML ). Ringraziamo Jozien Jaspers per il suo aiuto nel B-cell sorting mediante citometria di flusso.

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenge+of+identification+of+autoantibodies+specific+to+systemic+autoimmune+rheumatic+diseases+in+high+throughput+operation:+Proposal+of+reliable+and+feasible+strategies.">The challenge of identification of autoantibodies specific to systemic autoimmune rheumatic diseases in high throughput operation: Proposal of reliable and feasible strategies.</a> Clin Chim Acta. 437, 403-410 (2014).</li><li>Losen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+myasthenia+gravis+by+preventing+acetylcholine+receptor+modulation.">Treatment of myasthenia gravis by preventing acetylcholine receptor modulation.</a> Ann N Y Acad Sci. 1132, 174-179 (2008).</li><li>Smith, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+generation+of+fully+human+monoclonal+antibodies+specific+to+a+vaccinating+antigen.">Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen.</a> Nat Protoc. 4 (3), 372-384 (2009).</li><li>Fraussen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+making+human+monoclonal+antibodies.">A novel method for making human monoclonal antibodies.</a> J Autoimmun. 35 (2), 130-134 (2010).</li><li>Traggiai, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+to+make+human+monoclonal+antibodies+from+memory+B+cells:+potent+neutralization+of+SARS+coronavirus.">An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus.</a> Nat Med. 10 (8), 871-875 (2004).</li><li>Fraussen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantigen+induced+clonal+expansion+in+immortalized+B+cells+from+the+peripheral+blood+of+multiple+sclerosis+patients.">Autoantigen induced clonal expansion in immortalized B cells from the peripheral blood of multiple sclerosis patients.</a> J Neuroimmunol. 261 (1-2), 98-107 (2013).</li><li>Yamashita, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+individual+immune+responses+elicited+by+in+vitro+immunization.">Different individual immune responses elicited by in vitro immunization.</a> Cytotechnology. 40 (1-3), 161-165 (2002).</li><li>Hui-Yuen, J., Koganti, S., Bhaduri-McIntosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+B+cell+immortalization+for+monoclonal+antibody+production.">Human B cell immortalization for monoclonal antibody production.</a> Methods Mol Biol. 1131, 183-189 (2014).</li><li>Tiller, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+monoclonal+antibodies+from+single+human+B+cells+by+single+cell+RT-PCR+and+expression+vector+cloning.">Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning.</a> J Immunol Methods. 329 (1-2), 112-124 (2008).</li><li>Lanzavecchia, A., Corti, D., Sallusto, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+monoclonal+antibodies+by+immortalization+of+memory+B+cells.">Human monoclonal antibodies by immortalization of memory B cells.</a> Curr Opin Biotechnol. 18 (6), 523-528 (2007).</li><li>Traggiai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+human+B+cells:+analysis+of+B+cell+repertoire+and+production+of+human+monoclonal+antibodies.">Immortalization of human B cells: analysis of B cell repertoire and production of human monoclonal antibodies.</a> Methods Mol Biol. 901, 161-170 (2012).</li><li>Strober, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+cell+growth.+Curr+Protoc+Immunol+/+edited+by.">Monitoring cell growth. Curr Protoc Immunol / edited by.</a> John E. Coligan ... [et al.]. Appendix 3, Appendix 3A (2001).</li><li>Ibrahim, S. F., van den Engh, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+and+cell+sorting.">Flow cytometry and cell sorting.</a> Adv Biochem Eng Biotechnol. 106, 19-39 (2007).</li><li>Leith, J. T., Padfield, G., Faulkner, L. E., Quinn, P., Michelson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+feeder+cells+on+the+X-ray+sensitivity+of+human+colon+cancer+cells.">Effects of feeder cells on the X-ray sensitivity of human colon cancer cells.</a> Radiother Oncol. 21 (1), 53-59 (1991).</li><li>Hui-Yuen, J., McAllister, S., Koganti, S., Hill, E., Bhaduri-McIntosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+Epstein-Barr+virus+growth-transformed+lymphoblastoid+cell+lines.">Establishment of Epstein-Barr virus growth-transformed lymphoblastoid cell lines.</a> J Vis Exp. (57), 3321 (2011).</li><li>Smith, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+detection+in+automated+DNA+sequence+analysis.">Fluorescence detection in automated DNA sequence analysis.</a> Nature. 321 (6071), 674-679 (1986).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

In questo manoscritto, tutti i passaggi per la generazione di anticorpi IgG da PBMC umane sono presentati in dettaglio. Questo protocollo comprende alcuni vantaggi rispetto alle tecniche precedentemente pubblicati. Uno dei vantaggi è che gli anticorpi prodotti mantiene le catene pesanti e leggere corrispondenti alla coppia originale nel clone cellulare B. L&#39;identificazione di anticorpi IgG può essere fatto in qualsiasi tipo di donatore umano, e non vi è alcuna necessità di esacerbazione della risposta immunitari...

L&#39;obiettivo di questo articolo è quello di descrivere in dettaglio una metodologia per generare e caratterizzare gli anticorpi monoclonali IgG umane ottenute da cellule umane mononucleate del sangue periferico (PBMC). L&#39;interesse per lo studio anticorpi umani si è sviluppata in diversi campi di ricerca. In particolare, molti gruppi di ricerca sono interessati alla patologia causata da autoanticorpi 1-3. Abbiamo clonato e caratterizzato patogeni auto-anticorpi 1. Lo studio di auto-anticorpi può aiutare a identificare i loro obiettivi e per lo sviluppo di strategie terapeutiche, ad esempio, l&#39;uso di anticorpi concorrenti 4. Inoltre, lo studio di anticorpi umani può anche essere di interesse in altri campi di ricerca, cioè, per valutare la risposta immunitaria dopo la vaccinazione 5, per caratterizzare il profilo di anticorpi di individui che sono stati esposti e che sono diventate resistenti a patogeni specifici 6 o per studiare quale anticorpi sono inil repertorio naturali 7,12. Numerose tecniche sono state sviluppate per generare anticorpi monoclonali umani ricombinanti 8-12; la maggior parte di questi utilizzano phage display e immortalizzazione cellule B. L&#39;uso di phage display è stato ampiamente applicato per la scoperta di nuovi anticorpi 13. Tuttavia ha un grande svantaggio, vale a dire che le coppie di catena pesante e leggera della immunoglobulina umana diventare dissociato nel processo. La produzione di ibridomi con cellule B umane o trasformazione EBV supera questo inconveniente. Usiamo l&#39;infezione delle cellule B del timo con EBV in combinazione con la stimolazione delle cellule B policlonale via Toll-like receptor 9 (TLR-9) 6,12. In questo articolo, descriviamo in dettaglio la tecnologia che usiamo per lo sviluppo di anticorpi umani IgG, con una panoramica completa di tutte le fasi di isolamento PBMC alla vitro generazione di anticorpi in. Questoprotocollo può essere utilizzato per l&#39;analisi di qualsiasi tipo di profilo IgG umana. Nel nostro laboratorio, le cellule B che producono anticorpi IgG sono stati separati con successo dal resto del PBMC dopo la cernita. Cinquanta ordinati cellule B 8 possono essere placcati in piastre multi-pozzetti e immortalati da EBV e TLR-9 di attivazione, per l&#39;espansione clonale di singole cellule B. Come cellule feeder, sono stati utilizzati fibroblasti di tessuto polmonare embrionali, linea cellulare wi38, che facilita la visualizzazione delle cellule B immortalizzate. Da queste cellule B, le sequenze delle catene pesanti e leggere di immunoglobulina possono essere ottenuti mediante PCR, e geni gli anticorpi &#39;clonati in vettori di espressione immunoglobulina G e prodotti in vitro. Usando questa tecnica, singoli anticorpi con esattamente la stessa sequenza anticorpo trovato nel donatore possono essere studiate.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Histopaque-1077&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td>solution containing polysucrose and sodium diatrizoate</td>
		</tr>
		<tr>
			<td>FACSAria II cell sorter&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 U-bottom micro well plates&#160;</td>
			<td>Costar</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced Roswell Park Memorial Institute (RPMI) 1640 medium&#160;</td>
			<td>Gibco, Life Technologies</td>
			<td>12633-020</td>
			<td></td>
		</tr>
		<tr>
			<td>30% v/v EBV-containing supernatant of the B95-8 cell line&#160;&#160;</td>
			<td>ATCC</td>
			<td>CRL-1612</td>
			<td>3.4 x 108 copies/ml</td>
		</tr>
		<tr>
			<td>CpG2006&#160;</td>
			<td>Invivogen</td>
			<td>ODN 7909</td>
			<td></td>
		</tr>
		<tr>
			<td>Wi38 cells&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>90020107</td>
			<td></td>
		</tr>
		<tr>
			<td>Interleukin-2</td>
			<td>Roche&#160;</td>
			<td>10799068001</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA plates</td>
			<td>Greiner Bio-One, Microlon</td>
			<td>655092</td>
			<td></td>
		</tr>
		<tr>
			<td>AffiniPure F(ab&#39;)2 Fragment Goat Anti-Human IgG, Fc&#947; Fragment Specific (unconjugated)</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-006-008</td>
			<td></td>
		</tr>
		<tr>
			<td>4% non-fat dry milk (Blotting Grade Blocker)&#160;</td>
			<td>Biorad</td>
			<td>170-6404</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IgG&#160;</td>
			<td>Sigma</td>
			<td>I 2511</td>
			<td>HUMAN IgG purified Immunoglobulin, 5.6 mg/ml</td>
		</tr>
		<tr>
			<td>Goat F(ab)2 antihuman IgG Fc&#947; (conjugated with peroxidase (PO))</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-036-008</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA reader (Perkin Elmer 2030)&#160;</td>
			<td>Perkin Elmer&#160;</td>
			<td>2030-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase-conjugated AffiniPure Rabbit Anti-Human IgM, Fc5&#181;&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>309-035-095</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III Cells Direct cDNA Synthesis System&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>18080-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Applied Biosystems (ABI) GeneAm PCR System 2700</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High Pure RNA Isolation Kit&#160;</td>
			<td>Roche</td>
			<td>11828665001</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse transcription system kit&#160;</td>
			<td>Promega</td>
			<td>A3500</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Taq DNA Polymerase</td>
			<td>TAKARA</td>
			<td>R001A</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers (2&#956;l)</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure Agarose&#160;</td>
			<td>Invitrogen</td>
			<td>16500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>100 bp ladder</td>
			<td>Invitrogen</td>
			<td>15628-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantity One 4.5.2 (Gel Doc 2000)</td>
			<td>Biorad</td>
			<td>170-8100</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>QIAGEN</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr>
			<td>BigDye Terminator v3.1 cycle sequencing kit&#160;</td>
			<td>Applied Biosystems</td>
			<td>4337455</td>
			<td></td>
		</tr>
		<tr>
			<td>0.1 ml reaction plate (MicroAMP Optical 96-well)</td>
			<td>Applied Biosystems</td>
			<td>4346906</td>
			<td></td>
		</tr>
		<tr>
			<td>Genetic analyser ABI300&#160;</td>
			<td>Applied Biosystems</td>
			<td>4346906</td>
			<td></td>
		</tr>
		<tr>
			<td>DH5&#945; competent cells (E. coli)</td>
			<td>Invitrogen</td>
			<td>18263-012</td>
			<td></td>
		</tr>
		<tr>
			<td>pFUSEss-CHIg-hG1 (4493 bp)</td>
			<td>Invivogen</td>
			<td>pfusess-hchg1</td>
			<td></td>
		</tr>
		<tr>
			<td>pFUSEss-CHIg-hG4 (4484 bp)&#160;</td>
			<td>Invivogen</td>
			<td>pfusess-hchg4</td>
			<td></td>
		</tr>
		<tr>
			<td>pFUSE2ss-CLIg-hk (3875 bp)</td>
			<td>Invivogen</td>
			<td>pfuse2ss-hclk</td>
			<td></td>
		</tr>
		<tr>
			<td>pFUSE2ss-CLIg-hl2 (3883 bp)&#160;</td>
			<td>Invivogen</td>
			<td>pfuse2ss-hcll2</td>
			<td></td>
		</tr>
		<tr>
			<td>SOC medium</td>
			<td>Invitrogen</td>
			<td>15544-034</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-based agar medium supplemented with Zeocin (Fast-Media Zeo Agar)</td>
			<td>Invivogen</td>
			<td>fas-zn-s</td>
			<td></td>
		</tr>
		<tr>
			<td>Terrific Broth (TB)-based liquid medium supplemented with Zeocin (Fast-Media Zeo TB)</td>
			<td>Invivogen</td>
			<td>fas-zn-l</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Miniprep kit&#160;</td>
			<td>Omega Bio Technology</td>
			<td>D6942-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop (ND1000 Spectrophotometer)</td>
			<td>Nanodrop</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB-based agar medium supplemented with Blasticidin (Fast-Media Blast Agar)</td>
			<td>Invivogen</td>
			<td>fas-bl-s</td>
			<td></td>
		</tr>
		<tr>
			<td>Terrific Broth (TB)-based liquid medium supplemented with Blasticidin (Fast-Media Blast TB)</td>
			<td>Invivogen</td>
			<td>fas-bl-l</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI</td>
			<td>New England Biolabs</td>
			<td>R0101S</td>
			<td>20,000 U/ml, in 10x NEBuffer EcoRI</td>
		</tr>
		<tr>
			<td>NheI</td>
			<td>New England Biolabs</td>
			<td>R0131S</td>
			<td>10,000 U/ml, in 10x NEBuffer 2.1</td>
		</tr>
		<tr>
			<td>2-Log DNA ladder</td>
			<td>New England Biolabs</td>
			<td>N3200S</td>
			<td>0.1-10.0 kb, 1,000 &#956;g/ml</td>
		</tr>
		<tr>
			<td>XmaI</td>
			<td>New England Biolabs</td>
			<td>R0180S</td>
			<td>10,000 U/ml, in 10x CutSmart Buffer&#160;</td>
		</tr>
		<tr>
			<td>BsiWI</td>
			<td>New England Biolabs</td>
			<td>R0553S</td>
			<td>10,000 U/ml, in 10x NEBuffer 3.1</td>
		</tr>
		<tr>
			<td>AvrII</td>
			<td>New England Biolabs</td>
			<td>R0174S</td>
			<td>5,000 U/ml, in 10x CutSmart Buffer&#160;</td>
		</tr>
		<tr>
			<td>FastAP Thermosensitive Alkaline Phosphatase</td>
			<td>Thermo Scientific</td>
			<td>EF0651</td>
			<td>1 U/&#181;L, in 10x FastAP Buffer</td>
		</tr>
		<tr>
			<td>DH5&#945; competent cells&#160;</td>
			<td>Invitrogen</td>
			<td>18263-012</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse Anti-Human IgG</td>
			<td>BD Pharmingen</td>
			<td>555787</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD22, PerCP-Cy5.5, Clone: HIB22</td>
			<td>Fisher scientific</td>
			<td>BDB563942</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit&#160;</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>BigDye Terminator v3.1</td>
			<td>Applied Biosystems</td>
			<td>4337455</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of recombinant human igg monoclonal antibodies from immortalized sorted b cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Il gating ordinamento dopo colorazione cellule CD22 positive e IgG è mostrato in Figura 1 In questa immagine la zona delle cellule doppio positive -. È selezionata per ordinare tutte queste cellule in un tubo separato - cellule B che producono anticorpi IgG. Nell&#39;analisi, circa l&#39;1% dei PBMC totali corrispondono a questa doppia popolazione positiva. Il numero di cellule filtrate ottenute dipenderà dal numero di cellule ottenute nella sezione 1. I diversi risultati...

Watch this Scientific Journal Video about Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells at JoVE.com

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Generazione di ricombinante umano IgG anticorpi monoclonali da immortalizzate cellule ordinati B

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

generation-recombinant-human-igg-monoclonal-antibodies-from

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JoVE Journal

Immunology and Infection

18.2K Views.  Maastricht University. Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Video: Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Generazione di influenza da virus ricombinante del DNA plasmidi

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

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Immunologia e infezioni

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

Produzione ricombinante retrovirali e infezione delle cellule B

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating histoplasmosis caused by the fungus Histoplasma capsulatum and 2) to examine the role of virulence factors as target for therapy. To generate mAbs, mice are immunized, the immune responses are assessed using a solid phase ELISA system developed in our laboratory, and the best responder mice are selected for isolation of splenocytes for fusion with hybridoma cells. C57BL/6 mice have been extensively used to study H. capsulatum pathogenesis and provide the best model for obtaining the data required. In order to assess the role of the mAbs in infection, mice are intraperitoneally administered with either mAb to H. capsulatum or isotype matched control mAb and then infected by either intravenous (i.v.), intraperitoneal (i.p.), or intranasal (i.n.) routes. In the scientific literature, efficacy of mAbs for fungal infections in mice relies on mortality as an end point, in conjunction with colony formin units (CFU) assessments at earlier time points. Survival (time to death) studies are necessary as they best represent human disease. Thus, efficacy of our intervention would not adequately be established without survival curves. This is also true for establishing efficacy of vaccine or testing of mutants for virulence. With histoplasmosis, the mice often go from being energetic to dead over several hours. The capacity of an intervention such as the administration of a mAb may initially protect an animal from disease, but the disease can relapse which would not be realized in short CFU experiments. In addition to survival and fungal burden assays, we examine the inflammatory responses to infection (histology, cellular recruitment, cytokine responses). For survival/time to death experiments, the mice are infected and monitored at least twice daily for signs of morbidity. To assess fungal burden, histopathology, and cytokine responses, the mice are euthanized at various times after infection. Animal experiments are performed according to the guidelines of the Institute for Animal Studies of the Albert Einstein College of Medicine.

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

C57BL/6 mice have been used to study Hc pathogenesis and provide the best model. We are exploring the potential benefits of humoral immunity against this fungus and generated several mAbs [to histone H2B and a heat shock protein 60kDa] that we tested for their protective efficacy after intraperitoneal administration.

Amministrazione passivo di anticorpi monoclonali contro H. capsulatum E altri patogeni fungini

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Generazione di cellule T regolatrici indotte da cellule primarie umane T naive e memoria

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

Generazione di Arenavirus ricombinante per lo sviluppo del vaccino nel approvati dalla FDA cellule Vero

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Generazione di Human alloantigene-specifiche cellule T da sangue periferico

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Generazione di Murine anticorpi monoclonali da ibridomi Tecnologia

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Generazione di derivate da monociti cellule dendritiche umane da sangue intero

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Generazione di varianti di fuga di neutralizzare l'Influenza Virus anticorpi monoclonali

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Generazione di anticorpi monoclonali umani discriminante da rari B antigene-specifiche cellule circolanti nel sangue

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...